The broad objective of the proposed work is to better understand the role of oxidative stress in the development of human nuclear cataract, the most common type of lens opacity in older adults, and the type most likely to require surgery. The overall hypothesis to be tested is that both molecular oxygen (O2), as well as low molecular weight (LMW) crystallin fragments <3.5 kDa in size, contribute to the formation of high molecular weight (HMW) protein aggregates, causing increased nuclear light scatter and, eventually, nuclear cataract. A major focus is the use of the PI's well-established hyperbaric oxygen (HBO)/guinea pig in vivo model for nuclear cataract. Aim 1 will investigate the mechanism of O2-induced disulfide-crosslinking of lens crystallins, a modification strongly associated with human nuclear cataract. The hypothesis here is that crystallin glutathiolation (the binding of glutathione to a protein), but possibly not cysteinylation (the binding of cysteine, protects against disulfide-crosslinking and crystallin insolubilization in the HBO/guinea pig in vivo model. State-of-the-art mass spectrometry (MS) methods, including Multidimensional Protein Identification Technology and Electron-Transfer Dissociation, will be used to identify specific crystallin sites of glutathiolation and cysteinylation, and protein disulfide formation, respectively. Experiments are designed to identify which crystallins in the O2-stressed lens nucleus, and specifically which crystallin cysteine residues, are most likely to bind glutathione and cysteine, crosslink with other crystallins, and either stay in solution or form HMW aggregates and precipitate. In vitro experiments using isolated and recombinant A-, -, ?- and ?- crystallins will also be employed. Aim 2 will test the hypothesis that an elevation in the levelof O2 in the lens nucleus causes further truncation of crystallin fragments 6-12 kDa in size, formed in the nucleus as a result of normal aging, to LMW peptides <3.5 Da, which accelerate protein aggregation. The powerful technique of imaging mass spectrometry will be employed to visually follow the formation and disappearance of truncated crystallin fragments in various regions of thin tissue sections of guinea pig lenses, as a function of age and HBO- treatment. LC/MS analysis of control and HBO-treated guinea pig lens extracts will be employed to determine whether certain specific LMW peptides in the nucleus become associated with water-soluble HMW aggregates and water-insoluble protein as a result of O2-induced stress. In vitro experiments using recombinant human truncated A-crystallin fragments will be used to investigate the mechanism of O2-induced disappearance of the 6-12 kDa fragments in the lens nucleus. Impact: The studies are designed to elucidate the mechanism of maturity-onset nuclear cataract, as well as vitrectomy-induced nuclear cataract, which are major contributors to the 1.5 million cataract surgeries performed in the United States each year. Understanding mechanisms involved in aggregation of proteins in the aging human lens nucleus will aid in developing therapeutic agents to slow the process, and delay the onset of human nuclear cataract. PUBLIC HEALTH RELEVANCE: This application concerns the mechanism of formation of human nuclear cataract, the most common type of lens opacity in older adults, and the type most likely to require surgery. The proposed work will investigate factors leading to severe aggregation of proteins within the center of the lens, causing cataract. Understanding mechanisms involved in aggregation of proteins in the lens will aid in developing therapeutic agents to slow the process, and delay the onset of both maturity-onset and vitrectomy-induced nuclear cataract.